Multimarker Immunostaining of Neuronal and Glial Cells

Neurons were immunostained with a mouse monoclonal anti-NeuN antibody (1:200, MilliporeSigma, Carlsbad, CA, United States), astrocytes were detected by means of a polyclonal rabbit antibody anti-Glial Fibrillary Acidic Protein (GFAP, 1:500, DakoCytomation, Glostrup, Denmark), Cytochrome C with a mouse monoclonal antibody (1:200, Abcam, Cambridge, United Kingdom). Activated mTOR was detected using a polyclonal rabbit primary antibody raised against phospho-(Ser2448)-mTOR (1:100, Abcam, Cambridge, United Kingdom). Fluorescent secondary antibodies: Alexa Fluor 488 donkey anti rabbit (fluorescence in green, 1:400), Alexa Fluor 555 donkey anti mouse (fluorescence in red, 1:400), Alexa Fluor 635 goat anti-rabbit (fluorescence in far red, 1:400) (all from Life Technologies, Carlsbad, CA, United States). All primary and secondary antibodies were dissolved in Blocking Buffer (BB, 10% Normal Goat Serum, 0.05% NaN₃ in PBS-TX). All procedures were carried out with the free-floating method in wells of a 24-well plate (Cerbai et al., 2012 (link); Lana et al., 2013 (link)).

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Fusco I., Ugolini F., Lana D., Coppi E., Dettori I., Gaviano L., Nosi D., Cherchi F., Pedata F., Giovannini M.G, & Pugliese A.M. (2018). The Selective Antagonism of Adenosine A2B Receptors Reduces the Synaptic Failure and Neuronal Death Induced by Oxygen and Glucose Deprivation in Rat CA1 Hippocampus in Vitro. Frontiers in Pharmacology, 9, 399.

Publication 2018

anti-Glial Fibrillary Acidic Protein (GFAP, Alexa Fluor 488 donkey anti rabbit Alexa Fluor 555 donkey anti mouse

Alexa fluor 488 Alexa fluor 555 Anti glial fibrillary acidic protein antibody Antibodies Antibody Astrocytes Buffer Cytochrome c Donkey Fluorescence Fluorescent antibodies Gfap Goat Lana Monoclonal antibody Mouse Mtor Nan3 Neurons Rabbit Serum

Corresponding Organization : University of Florence

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Variable analysis

independent variables

Mouse monoclonal anti-NeuN antibody (1:200, MilliporeSigma, Carlsbad, CA, United States)
Polyclonal rabbit antibody anti-Glial Fibrillary Acidic Protein (GFAP, 1:500, DakoCytomation, Glostrup, Denmark)
Mouse monoclonal antibody against Cytochrome C (1:200, Abcam, Cambridge, United Kingdom)
Polyclonal rabbit primary antibody raised against phospho-(Ser2448)-mTOR (1:100, Abcam, Cambridge, United Kingdom)

dependent variables

Neurons
Astrocytes
Cytochrome C
Activated mTOR

control variables

Blocking Buffer (BB, 10% Normal Goat Serum, 0.05% NaN3 in PBS-TX)
Free-floating method in wells of a 24-well plate

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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